c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.


Minor Comments:
• The authors propose an interactive model between c-Myc, Cul4b, p21, and Cyclin E2.It would be beneficial to provide a summary diagram that highlights these interactions in the WT and KO conditions.
• Additional citations within the introduction and results would be helpful in lines 57, 73, 77, 107.
• A description of the c-Myc conditional knock-out mouse model is missing from the methods section.
• In line 322, the authors state that the number of P14 cells is decreased but this should state frequency since enumeration of cells was not shown for this data.
• The figures would benefit from using open/closed circles or another strategy to distinguish between experimental groups in graphs.When printed in black and white, it is very difficult to tell which group is which in the figures (see Figure 2B, 4B, Figure 5I as examples).
• Figure 1A needs a scale for understanding the significance of bubble size.
• Figure 2A displays overlapping flow cytometry gates.Please adjust these so that events are not being counted within both populations.
• Figure 6A western blotting analysis would benefit from quantitative analysis and/or specification of repeat blots within the supplemental figures.• Figure 6C only has two sizes listed for P-values but there are three sizes within the plot.Please clarify what the smallest dot size represents.
• Please include the clones and manufacturer of the antibodies used in the naive CD8 T cell isolation (lines 599 -600) Reviewer #2 (expert in T cell memory): This is a very well-executed study showing a striking failure of T cell population expansion upon TCR triggering in Cul4b deficient hosts.It establishes that the central driver of T cell proliferation, c-Myc, increases expression of Cul4b, which then localizes to the chromatin to limit the accumulation of proteins implicated in replication stress.While the authors have shown some of these effects already in the context of CD4 T cell activation, sufficient novelty persists due to the extensive in vivo data (infection models, effects in viral titres, mouse survival etc.) presented and the link to c-Myc that is established for the first time.
I would like the authors to address three more points to provide a fully rounded manuscript: 1) Does the observed replication stress in Cul4b deficient CD8 T cells limit T cell expansion upon (e.g.LCMV infection) via restriction of proliferation and/or increased induction of apoptosis?2) Is PD-1 upregulation and T cell exhaustion a direct consequence of replication stress or induced due to limited population expansion and the resulting failure to clear LCMV-Arm infection.This could be answered by investigating PD-1 expression and cytokine production in Cul4b deficient P14 T cells transferred to WT hosts and subsequently infected with LCMV-Arm.In this setting virus will be controlled by the endogenous T cells excluding viral persistence as a potential culprit.
3) The manuscript should be shortened and sharpened.It feels a bit encyclopaedic and at times looses focus with respect to the authors' key messages.

Reviewer #3 (expert in cell cycle regulation):
This study identifies a role for Cul4b downstream of c-Myc as a regulator of cell proliferation in CD8+ T cells.The study shows that Cul4b is important for activation-induced and homeostatic proliferation of CD8+ T cells.Strikingly, condition deletion of Cul4b in CD4+ expressing cells results in defective memory responses to LCMV in vivo.Mechanistically, Cul4b deletion results in increased DNA damage and genome instability in proliferating CD8+ T cells, leading to arrest/delays in G2&M phases of the cell cycle and presumably cell death.It remains unclear why Cul4b increases DNA damage in CD8+ T cells, although a role for Cyclin E2 and p21 overexpression are implicated.Notably, p21 is highly induced following TCR activation (e.g., http://www.immpres.co.uk/), but the absolute levels are important for the function of p21 as a CDK inhibitor protein, and Cul4bcKO appears to produce even higher levels of p21.These are important findings that will be of interest to the field.

Major points:
The manuscript requires a significant re-write as there are grammatical issues throughout that make interpretation of the study challenging.

Does Cul4bcKO affect cell survival?
Is there a difference in cell size in the activated MyccKO and Cul4bcKO CD8+ T cells compared to WT?
Is there re-replication in the activated Cul4bcKO CD8+ T cells?

Reviewer #1 (expert in T cell memory):
This manuscript builds upon previous work from the Oliver lab invesfigafing the role of Cul4b regulafion of DNA damage response in CD4 T cells.Here the authors idenfify a role for Cul4b in CD8 T cell proliferafion, effector response, and memory formafion.Through transcriptomic, proteomic, and cellular immunologic methods, the authors idenfify a regulatory network where c-Myc upregulates Cul4b in T cells to mifigate acfivafion-induced DNA damage and cell cycle entrance through regulafion of p21 and Cyclin E2.This manuscript provides insight into the molecular mechanisms of managing T cell replicafive stress.While this manuscript provides new insight into the interacfions between c-Myc, Cul4b, p21, and Cyclin E2, there are a few concerns regarding the model and molecular mechanisms of these interacfions.

Major Comments:
• Data supporfing normal thymic development of the Cul4b-cKO animals would be important to demonstrate that the Cul4b naïve T cells are comparable to WT cells prior to acfivafion.Enumerafion of thymic developmental stages as well as repertoire analysis would be beneficial.In the Dar AA 2021 PLoS Biology publicafion, there were data to suggest decreased CD8 T cells in the thymus and spleen of Cul4b-cKO mice which may impact the interpretafions of the peripheral T cell responses examined within this manuscript.An inducible knock-out system may complement or improve the design of these experiments to determine the contribufion of Cul4b in peripheral T cell responses and remove any confounding variables from the proliferafive burst during thymic development.
Answer: This is an important point.Cul4, substrate receptor DCAF1 has been shown to interact with RAG and regulate its expression hence influence the TCR rearrangement (Kassmeier, Mondal et al. 2012) (Schabla, Perry et al. 2018) .In this model, Cul4b is very likely deleted after CD4 is expressed, and thus after TCR rearrangement.Nonetheless, it is important for us show that the TCR repertoire is not impacted.In our previous study we reported that there was a marginal decrease in CD8 + T cell percentages in the thymus and periphery however these differences were not seen when we assessed cell numbers.To assess this in greater detail, we performed flow cytometric analysis of the TCR Vβ repertoire in thymic T cells from control and Cul4b cKO mice.The usage of several Vβ chains in Cul4b deficient T cells was compared to the control cells.We did not observe any significant impact of TCR Vβ chain usage for any chains tested.TCR repertoire data from thymic SP CD8 + T cells is now shown in the supplementary figure 2l.The similarifies of TCR Vβ repertoire between these control mice and Cul4 cKO mice indicates that Cul4b delefion at the DP stage did not change the TCR repertoire and hence is unlikely to impact the outcome of current study.The changes made to the text are highlighted in yellow at line 211-213.
• The authors invesfigate the impairment of the DNA damage response in Cul4b-cKO cells but, it would be helpful to use an addifional funcfional readout like a Comet assay that was used in the Dar AA et al paper.The addifion of this assay would complement other data in Figure 7 of this manuscript to strengthen the evidence of increased DNA damage in Cul4b-cKO CD8 T cells.
Answer: As suggested by the reviewer, we performed a comet assay to access the extent of the DNA damage in acfivated control and Cul4b cKO CD8 + T cells after Camptothecin treatment.The comet assay revealed that Cul4b cKo CD8 + T cells showed more DNA damage than their control counterparts.The analyzed data is now included in the figure 7d,e and necessary changes are made in the text and highlighted in yellow at line 398-401, 600-610.Overall, our data provides strong evidence that CD8 + T cells lacking Cul4b have increased DNA damage.

Minor Comments:
• The authors propose an interacfive model between c-Myc, Cul4b, p21, and Cyclin E2.It would be beneficial to provide a summary diagram that highlights these interacfions in the WT and KO condifions.
Answer: As requested by the reviewer we have included a pathway diagram to summarize our findings.The pathway diagram is included as supplementary figure 9i • Addifional citafions within the introducfion and results would be helpful in lines 57, 73, 77, 107.
Answer: As suggested, we have included the relevant citafions • A descripfion of the c-Myc condifional knock-out mouse model is missing from the methods secfion.Answer: We would like to clarify that we did not maintain the c-Myc mice colony, the data on c-Myc was analyzed from the publicly available datasets.For each dataset the relevant source was cited in the results and method secfion.For example, line 119, 715, 742, 760,767.However, at the places where we missed cifing the source, we have cited it in the revised manuscript for example line 391.
• In line 306, the marker is listed as 'CXC3CR1' instead of 'CX3CR1'.Answer: Thanks for poinfing out this we corrected the typo • In line 322, the authors state that the number of P14 cells is decreased but this should state frequency since enumerafion of cells was not shown for this data.Answer: We thank reviewer for noficing this.As the control and Cul4b cKO P14 cells were maintained in the same recipient mice, the decrease in percentages would be inevitably reflected at numbers.However, for clarity we replaced numbers with percentages at line 306 in revised manuscript.
• The figures would benefit from using open/closed circles or another strategy to disfinguish between experimental groups in graphs.When printed in black and white, it is very difficult to tell which group is which in the figures (see Figure 2B, 4B, Figure 5I as examples).
Answer: We used the strategy of represenfing data points with open circles.The control group is represented in black circle while Cul4b cKO group in burgundy red.For the sake of convenience, we have modified the line graphs for example figure 2b, 3b, 4g, h while the bar graphs have been labelled wherever labeling was missing for example 5b, 5d, 5f, 2k, etc • Figure 1A needs a scale for understanding the significance of bubble size.Answer: The size or area of the displayed circles is proporfional to the number of genes assigned to the term.The statement is included in the figure legend and also figures have been updated in the revised manuscript • Figure 2A displays overlapping flow cytometry gates.Please adjust these so that events are not being counted within both populafions.Answer: As suggested, we have adjusted the gates • Figure 6A western blofting analysis would benefit from quanfitafive analysis and/or specificafion of repeat blots within the supplemental figures.Answer: The replicates of the experiment will be included in the source data

Reviewer #2 (expert in T cell memory):
This is a very well-executed study showing a striking failure of T cell populafion expansion upon TCR triggering in Cul4b deficient hosts.It establishes that the central driver of T cell proliferafion, c-Myc, increases expression of Cul4b, which then localizes to the chromafin to limit the accumulafion of proteins implicated in replicafion stress.While the authors have shown some of these effects already in the context of CD4 T cell acfivafion, sufficient novelty persists due to the extensive in vivo data (infecfion models, effects in viral fitres, mouse survival etc.) presented and the link to c-Myc that is established for the first fime.
I would like the authors to address three more points to provide a fully rounded manuscript: 1) Does the observed replicafion stress in Cul4b deficient CD8 T cells limit T cell expansion upon (e.g.LCMV infecfion) via restricfion of proliferafion and/or increased inducfion of apoptosis?Answer: This is an important clarificafion.As we have shown that Cul4b-deficient CD8 + T cells proliferated and expanded less than control cells as evidenced in Figure 2A-D.The reduced expansion of Cul4b-deficient CD8 + T cells could also be an indicafion of their poor survival rate.To test this, we sfimulated cells under in vitro culture condifions and analyzed cell death using Annexin V staining.We found significantly higher percentages of Cul4b-deficient CD8 + T cells undergoing apoptosis (fig 7k and supplementary fig.9f).However, when cell proliferafion was blocked using rapamycin the control and Cul4b-deficient CD8 + T did not show any difference in cell death (supplementary fig.9g, h).This Indicates that in absence of Cul4b, CD8 + T cells are not able to circumvent the replicafion stress hence proliferate less and also undergo apoptofic death.
2) Is PD-1 upregulafion and T cell exhausfion a direct consequence of replicafion stress or induced due to limited populafion expansion and the resulfing failure to clear LCMV-Arm infecfion.This could be answered by invesfigafing PD-1 expression and cytokine producfion in Cul4b deficient P14 T cells transferred to WT hosts and subsequently infected with LCMV-Arm.In this sefting virus will be controlled by the endogenous T cells excluding viral persistence as a potenfial culprit.
Answer: We thank reviewer for making this point.As suggested, we transferred the control and Cul4b cKO P14 T cells into a congenically disfinct WT mice.The recipient cells were analyzed for the PD-1 expression at day 30 post infecfion.Neither donor cells (control and Cul4b cKO P14 T) or recipient cells expressed PD-1.This clearly indicates that the increased PD-1 expression found on Cul4b-deficient CD8 + T cells was due to their inability to clear the virus.The data is included in the supplementary figure 6J and this is now clarified in the text.The ability to produce cytokines is dependent on Cul4b in a cell intrinsic fashion as shown in figure 2l and Supplementary I,j,k).
3) The manuscript should be shortened and sharpened.It feels a bit encyclopaedic and at fimes looses focus with respect to the authors' key messages.
Answer: We have aftempted to reduce the text to make the manuscript more concise.
Reviewer #3 (expert in cell cycle regulafion): This study idenfifies a role for Cul4b downstream of c-Myc as a regulator of cell proliferafion in CD8+ T cells.The study shows that Cul4b is important for acfivafion-induced and homeostafic proliferafion of CD8+ T cells.Strikingly, condifion delefion of Cul4b in CD4+ expressing cells results in defecfive memory responses to LCMV in vivo.Mechanisfically, Cul4b delefion results in increased DNA damage and genome instability in proliferafing CD8+ T cells, leading to arrest/delays in G2&M phases of the cell cycle and presumably cell death.It remains unclear why Cul4b increases DNA damage in CD8+ T cells, although a role for Cyclin E2 and p21 overexpression are implicated.Notably, p21 is highly induced following TCR acfivafion (e.g., hftp://www.immpres.co.uk/), but the absolute levels are important for the funcfion of p21 as a CDK inhibitor protein, and Cul4bcKO appears to produce even higher levels of p21.These are important findings that will be of interest to the field.

Major points:
The manuscript requires a significant re-write as there are grammafical issues throughout that make interpretafion of the study challenging.
We have proof read the manuscript for grammafical issues-addifional changes will likely be made during the editorial process Does Cul4b cKO affect cell survival?
Answer: We would like to thank the reviewer for the comment.To confirm this, we sfimulated both control and Cul4b deleted CD8 + T cells in vitro, allowed them to proliferate for three days, and then used Annexin V staining to examine cell death.We found significantly higher percentages of Cul4b-deficient CD8 + T cells undergoing apoptosis (Figure 7k and Supplementary 9f).The data indicates that Cul4b-deficient CD8 + T cells survive less.
Is there a difference in cell size in the acfivated MyccKO and Cul4bcKO CD8+ T cells compared to WT? Answer: The difference in the cell size of MYC cKO compared to WT cells is already established (Marchingo, Sinclair et al. 2020).Myc-deficient T cells do not substanfially increase cell size or proliferate in response to immune acfivafion with anfi-CD3/anfi-CD28 agonist anfibodies.However, we did not find any difference in the size of surviving Cul4b cKo CD8 + T cells compared to WT. Forward scafter area (FSC-A) of anfi-CD3 + anfi-CD28 (TCR) acfivated control and Cul4b deficient CD8 + T cells is shown in the Supplementary Fig. 1i.This is stated in the revised manuscript line 166-167 Is there re-replicafion in the acfivated Cul4b cKO CD8 + T cells?Answer: To check whether Cul4b deficiency induces re-replicafion, the DNA content of control and Cul4b cKo CD8 + T cells was analyzed.Cul4b-deficient CD8 + T cells showed significantly increased numbers of cells with DNA >4N at day 2 after acfivafion.These data support rereplicafion (Fig. 7j and Supplementary Fig. 9e).At later fime point, day 3, this was associated with increased cell death as shown in Figure 7k and Supplementary Fig. 9f).These results suggest that re-replicafion induced by a lack of Cul4b ulfimately results in apoptosis in CD8 + T cells Figure 6C only has two sizes listed for P-values but there are three sizes within the plot.Please clarify what the smallest dot size represents.Answer: The p values corresponding to the smallest circle has been included in the figure • Please include the clones and manufacturer of the anfibodies used in the naive CD8 T cell isolafion (lines 599 -600) Answer: The anfibodies are provided in the kit (MACS Miltenyi Kit) as indicated in the line 583.The manufacturer does not disclose the clone of the anfibodies.